Thefiloviruses(EbolaandMarburg)causeseverehemorrhagicfeversyndromeswithhighmortalityrates.As these RNA viruses are classified as NIAID Category A pathogens, the current lack of effective vaccines and antiviral therapeutics for these deadly pathogens is particularly concerning. We and others have established thatefficientfilovirusbuddingiscriticallydependentonthesubversionofhostproteinsTsg101andNedd4and that viral PTAP and PPxY late (L) budding domains are critical for these interactions, respectively. As disruption of virus budding would prevent virus dissemination, we propose to directly evaluate the ability of small molecule inhibitors to disrupt Tsg101PTAP and Nedd4PPxY interactions, thereby preventing virus budding. Our collaborators, Drs. Michael Lee and Mark Olson (USAMRIID, Ft. Detrick, MD), have used the known structures of Tsg101PTAP and Nedd4PPxY interactions to guide the in silico selection/design of competitiveinteractionblockers.Wearecurrentlyevaluatingtheabilityoftopcandidateinhibitorstodisruptthe hostdependent egress of virus particles in viruslike particle (VLP) budding assays. As Tsg101PTAP and Nedd4PPxYinteractionsaretooweakand/ortransienttobedetectedbystandardbiochemicalapproaches, ourlaboratoryhassuccessfullydevelopedapowerfulbimolecularcomplementation(BiMC)approachtodetect theseviralhostinteractionsinlivecells.Wewillusethisinnovativetechniquetodeterminewhethercandidate inhibitors disrupt these virushost interactions. Promising preliminary results with one lead compound (5539 0062)supportthefeasibilityofthisproposal.AsLdomaincontainingmatrixproteinsarerequiredforefficient viruscell separation of many RNA viruses, including retroviruses, arenaviruses, rhabdoviruses, paramyxoviruses,henipaviruses,andfiloviruses,wepredictthattargetingtheinteractiondomainbetween filovirusVP40andhostTsg101andNedd4willserveasbasisforthedevelopmentofnewandpowerfulbroad spectrumantiviraldrugs.Ourgroupofhighlyinteractiveandexpertcollaboratorswillbuilduponourpromising preliminary results obtained following an in silico screen of 4.8 million compounds to functionally validate candidatesmallmoleculesthatinhibitfilovirusTsg101(Aim1)andfilovirusNedd4(Aim2)interactionsduring theR21phaseofthisproposal.IntheR33phaseofthisproposal,wewilldevelopandutilizeFluorescence Lifetime Imaging Microscopy (FLIM) to functionally/mechanistically categorize inhibitors as they disrupt virus host protein interactions in real time and assess their mode of action, thereby laying the foundation for developmentandtestingofmultidrugtherapies(Aim3).Lastly,wewilldeterminetheefficacyofinhibitorsin preclinicallivevirusstudiesusingWTandmutantVP40LdomainVSVrecombinantsalreadygenerated(BSL 2 setting). Moreover, in collaboration with Dr. G. Olinger (USAMRIID), we will test mature lead candidate inhibitorsfortheirabilitytoblockegressoflive,EBOVandMARVinaBSL4setting(Aim4).